Effects of human immunodeficiency virus type 1 nef and tat genes on rat PC12 pheochromocytoma cells

The regulatory genes nef and tat of the human immunodeficiency virus type 1 (HIV-1) were transferred into the rat pheochromocytoma cells (line PC12) under the control of the eukaryotic promoters. Proliferative activity of the PC12 cells transfected with the tat HIV-1 gene was substantially increased as compared to the control. Conversely, the nef gene introduced into the cultivated PC12 cell caused inhibition of their proliferative activity and formation of cell agglomerates resembling in morphology the multinuclear syncytial cells. Thus, our results suggest that the tat gene activates proliferation of the cultivated PC12 cells, whereas the nef gene inhibits proliferation of the same cells. We have obtained for the first time a direct indication for the possible role of the nef gene in formation of multinuclear T-lymphocyte and macrophage syncytium in HIV-1-infected patients. The HIV-1 nef and tat genes had no significant effect on the neuronal differentiation of the PC12 cells induced by the nerve growth factor (NGF).     

表达HIV-1六种基因的非复制型重组痘苗病毒在CEF细胞中遗传稳定

本研究目的是了解表达HIV-1六种基因的非复制型重组痘苗病毒(rNTV-C)的遗传稳定性(包括病毒载体和六种外源基因:gp160、gag、polr、evt、at和nef)。我们将rNTV-C在原代鸡胚成纤维细胞(CEF)中连续传代至25代,对第9、12、15以及25代病毒载体基因的稳定性、六种外源基因的稳定性、外源基因表达的稳定性以及外源基因的丢失率进行研究。结果显示:各代病毒均保持了非复制型天坛株痘苗病毒载体特点且传代稳定;HIV-1目的基因序列与原设计序列相符、重组位点正确且遗传稳定,连续传25代核苷酸突变率低于万分之一;目的蛋白在各代rNTV-C中均能有效表达,而且各代病毒之间表达量和分子量无明显差别;以Gag和Nef蛋白表达为标记,rNTV-C各代病毒的基因丢失率均在5%以下。本研究结果为疫苗生产提供了确保毒种稳定的关键资料。     

Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Immune responses in asymptomatic HIV-1-infected patients after HIV-DNA immunization followed by highly active antiretroviral treatment.

Intensive chemotherapy is capable of reducing the viral load in HIV-1-infected individuals while infected cells are still present. A special property of DNA immunization is to induce both new CTL and Ab responses. We evaluated the possibility of inducing new immune responses in already infected individuals by means of DNA constructs encoding the nef, rev, or tat regulatory HIV-1 genes. Significant changes in viral loads and CD4+ counts were observed in four patients who started highly active antiretroviral treatment (HAART) during the immunization study. The DNA immunization induced Ag-specific T cell proliferation, which persisted up to 9 mo after the last DNA injection, and cytolytic activities but did not, by itself, reduce viral load. Increased levels of CTL precursor cells were induced in all nine DNA-immunized patients. The profile of IFN-gamma secretion observed when human PBMC were transfected with the nef, rev, and tat DNA resembled that found in the CTL activity (nef > tat > rev). Ab responses that occurred after immunizations were of a low magnitude. In accordance with the high IL-6 production induced by the nef DNA plasmid, IgG titers were highest in patients immunized with nef DNA. The initiation of HAART appears to contribute to the induction of new HIV-specific CTL responses, but by itself did not cause obvious re-induction of these activities.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.     

Enhanced CD4 down-modulation by late stage HIV-1 nef alleles is associated with increased Env incorporation and viral replication

Three viral proteins participate in the down-modulation of CD4 in human immunodeficiency virus type 1 (HIV-1)-infected cells. The underlying mechanisms have been extensively investigated. However, the physiological relevance of this phenomenon remains poorly understood. To address the role of CD4 down-modulation in HIV-1 pathogenesis in vivo, we have characterized the functional properties of nef alleles isolated from seven HIV-1-infected patients at either the stage of AIDS (late alleles) or during the asymptomatic phase of infection (early alleles). HIV-1 variants carrying these nef alleles showed striking differences in CD4 down-modulation, virus infectivity, and replication properties. Infection of T cells with late strains resulted in production of viral particles with enhanced infectivity, as compared with variants carrying early nef alleles. These differences in infectivity were observed only when viruses were produced in cells with high levels of the viral receptor, suggesting a functional link between CD4 levels and the ability of Nef to down-modulate CD4 and to enhance viral infectivity. Similarly, late nef alleles were substantially more active than early nef genes in stimulating HIV-1 replication in high CD4-positive cells, including primary lymphocytes, but not in cells expressing low levels of the CD4 receptor. Single-round assays showed that differences in infectivity between late and early strains are largely reduced when evaluated in target cells with high levels of CD4, suggesting that the inhibitory effect occurs at the entry step. Supporting this, enhanced CD4 down-modulation by late nef alleles was associated with higher levels of envelope incorporation into viral particles, a phenomenon that likely accounted for the augmented infectivity. Our data suggest a mechanistic link between the Nef-mediated CD4 down-modulation and the enhancement of replication in CD4-positive lymphocytes. As progression to disease occurs, HIV-1 Nef variants with enhanced ability to down-modulate CD4 are selected. These strains efficiently overcome the deleterious effects of CD4 and replicate more aggressively in CD4-positive primary lymphocytes. These results highlight the importance of the virus-induced CD4 down-modulation in HIV-1 pathogenesis.     

5个HIV基因修饰可明显提高其在不同系统中的表达

目的:确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原,提高各个基因在相应疫苗载体中的表达水平,为研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定实验基础。方法:选择HIV B′/C亚型5个以细胞免疫为主的抗原(Gag、Pol、Rev、Tat和Nef),进行基因序列优化及表达结构改造,并分别构建以质粒DNA和重组痘苗病毒为载体的两大类HIV-1疫苗。结果:优化前后5个目的基因均能够在这2种载体中有效表达;虽然采用相同的基因修饰策略,但与痘苗病毒载体相比,在DNA载体中各基因表达水平的提高均较为明显;含有抑制性序列(INS)的gag、pol基因经密码子优化后,Gag、Pol蛋白的表达均明显提高,其中Pol蛋白的提高更为明显,单独pol基因比gagpol天然结构表达水平要高,而gag基因却变化不大;对于rev、tat、nef基因而言,优化后的单独基因结构要略高于优化后的融合结构(hRTN),且二者均高于未优化的融合结构(RTN)。结论:为进一步确定HIV-1疫苗中有效的交叉保护性细胞免疫抗原、研究不同抗原在DNA载体和痘苗病毒载体中的免疫原性奠定了实验基础,为进一步研究DNA疫苗和重组痘苗病毒疫苗联合免疫提供了实验依据。     

Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu, env, and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison, Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences, Nef inhibited superinfection more efficiently than Vpu and Env. Notably, nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus, maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally, we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly, one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production.     

Defective accessory genes in a human immunodeficiency virus type 1-infected long-term survivor lacking recoverable virus.

We have been studying a patient who acquired human immunodeficiency virus (HIV) infection via a blood transfusion 13 years ago. She has remained asymptomatic since that time. The blood donor and two other recipients have all died of AIDS. Although this patient has shown persistently strong seroreactivity to HIV type 1 (HIV-1) antigens by Western blot (immunoblot), she has been continually HIV culture negative in results from multiple laboratories over the last 6 years and has a very low viral burden. Her CD4+ T-cell count has fluctuated around a mean of 399 cells per microliters, with little change in lymphocyte subset percentages. Strong cellular immune responses to HIV-1 epitopes by this patient have been demonstrated. We now report the results of an intensive molecular genetic analysis of the HIV-1 proviral quasispecies from this patient sampled over 5 years. Long terminal repeat region sequences supported the argument for normal basal and Tat-mediated promoter activities. Sequential sequencing of the nef gene revealed a low frequency (8.3%) of defective genes and a striking lack of sequence evolution. Functional analysis of predominant nef genes by both a cell surface CD4 downregulation and a viral infectivity complementation assay showed wild-type function. In contrast, sequential analysis of an amplicon containing the vif, vpr, vpu, tat1, and rev1 genes revealed the presence of inactivating mutations in 64% of the clones. These data suggest that this patient, initially infected with a virulent swarm of HIV-1, is presently infected with a more-attenuated viral quasispecies as a result of effective host immunity.     

Evolution of the primate lentiviruses: evidence from vpx and vpr.

The genomes of the four primate lentiviral groups are complex and contain several regulatory or accessory genes. Two of these genes, vpr and vpx, are found in various combinations within the four groups and encode proteins whose functions have yet to be elucidated. Comparison of the encoded protein sequences suggests that the vpx gene within the HIV-2 group arose by the duplication of an ancestral vpr gene within this group. Evolutionary distance analysis showed that both genes were well conserved when compared with viral regulatory genes, and indicated that the duplication occurred at approximately the same time as the HIV-2 group and the other primate lentivirus groups diverged from a common ancestor. Furthermore, although the SIVagm vpx proteins are homologous to the HIV-2 group vpx proteins, there are insufficient grounds from sequence analysis for classifying them as vpx proteins. Because of their similarity to the vpr proteins of other groups, we suggest reclassifying the SIVagm vpx gene as a vpr gene. This creates a simpler and more uniform picture of the genomic organization of the primate lentiviruses and allows the genomic organization of their common precursor to be defined; it probably contained five accessory genes: tat, rev, vif, nef and vpr.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

Induction of AIDS in Rhesus Monkeys by a Recombinant Simian Immunodeficiency Virus Expressing nef of Human Immunodeficiency Virus Type 1

A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.     

cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4 intracytoplasmic tails

F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurring nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIV nef allele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef gene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi and nef-defective (Deltanef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.